Vaccination with inactivated or attenuated organisms or their products has been shown to be an effective method for increasing host resistance and ultimately has led to the eradication of certain common and serious infectious diseases. The use of vaccines is based on the stimulation of specific immune responses within a host. The use of vaccination to successfully prevent certain diseases, most notably small pox and poliomyelitis, represents a great triumph of immunology.
Unfortunately, effective vaccines have been developed for relatively few of the infectious agents that cause disease in domestic animals and man. This reflects technical problems associated with the growth and attenuation of virulent strains of pathogens. Recently, effort has been placed on the development of subunit vaccines (vaccines that present only selected antigens from a pathogen to the host). Subunit vaccines have the potential for achieving high levels of protection in the virtual absence of side effects. Subunit vaccines also offer the opportunity for the development of vaccines that are stable, easy to administer, and sufficiently cost-effective for widespread distribution.
One particular type of subunit vaccine contains a DNA vector that encodes a specific viral protein. DNA vaccines are described in copending U.S. Ser. No. 08/187,879 filed Jan. 27, 1994, and its corresponding Published International Application WO 95/20660; copending U.S. Ser. No. 08/009,833, filed Jan. 27, 1993, and copending U.S. Ser. No. 07/855,562, filed Mar. 23, 1992, and their corresponding Published International Application WO 93/19183, all disclosures of which are hereby incorporated by reference in their entireties.
There are numerous advantages of the use of such DNA vectors for immunizations. For example, immunization can be accomplished using any antigen encoded by DNA. Furthermore, the DNA encoded antigens are expressed as "pure" antigens in their native states and have undergone normal host cell modifications.
DNA is easily and inexpensively manipulated, and is stable over a wide range of temperatures either as a dry product or in solution. This technology is valuable not only for the development of vaccines against practically any agent, but furthermore can be used to manipulate the immune response in such varied conditions as cancer or during organ transplantation.
The ability of directly injected DNA, that encodes a viral protein, to elicit a protective immune response has been demonstrated in numerous experimental systems Conry et al., Cancer Res., 54:1164-1168 (1994)!, Cox et al., Virol, 67:5664-5667 (1993)!, Davis et al., Hum. Mole. Genet., 2:1847-1851 (1993)!, Sedegah et al., Proc. Natl. Acad. Sci., 91:9866-9870 (1994)!, Montgomery et al., DNA Cell Bio., 12:777-783 (1993)!, Ulmer et al., Science, 259:1745-1749 (1993)!, Wang et al., Proc. Natl. Acad. Sci., 90:4156-4160 (1993)!, Xiang et al., Virology, 199:132-140 (1994)!. Studies to assess this strategy in neutralization of influenza virus have used both envelope and internal viral proteins to induce the production of antibodies, but in particular have focused on the viral hemagglutinin protein (HA) Fynan et al., DNA Cell. Biol., 12:785-789 (1993A)!, Fynan et al., Proc. Natl. Acad. Sci., 90:11478-11482 (1993B)!, Robinson et al., Vaccine, 11:957, (1993)!, Webster et al., Vaccine, 12:1495-1498 (1994)!. The viral hemagglutinin protein is a glycoprotein that mediates adsorption and penetration of influenza virus, and is a major target for host neutralizing antibodies. Influenza virus hemagglutinin proteins exhibit fifteen different serological subtypes, HA 1 to HA 15, associated with the fifteen viral subtypes H1-H15 respectively.
Vaccination through directly injecting DNA, that encodes a viral protein, to elicit a protective immune response produces both cell-mediated and humoral responses. This is analogous to results obtained with live viruses Raz et al., Proc. Natl. Acad. Sci., 91:9519-9523 (1994)!, (Ulmer, 1993, supra), (Wang, 1993, supra), (Xiang, 1994, supra). Studies with ferrets indicate that DNA vaccines against conserved internal viral proteins of influenza, together with surface glycoproteins, are more effective against antigenic variants of influenza virus than are either inactivated or subvirion vaccines Donnelly et al., Nat.Medicine, 6:583-587 (1995)!. Indeed, reproducible immune responses to DNA encoding nucleoprotein have been reported in mice that last essentially for the lifetime of the animal Yankauckas et al., DNA Cell Biol., 12: 771-776 (1993)!.
The possibility of species-specific differences in responsiveness to DNA-based vaccines was first raised in studies by Robinson et al. in 1993, showing that direct inoculation of a defective retroviral vector expressing H7 HA could protect chickens against lethal H7 influenza virus challenge (Robinson, 1993, supra). These investigators reported a wide range of protection rates (Fynan, 1993B, supra), (Robinson, 1993, supra), which were on average considerably lower than the generally high responsiveness observed for mice (Fynan, 1993B, supra). These results show that results obtained for experimental inbred mice cannot necessarily be extrapolated to outbred chickens.
Variable results reported by Robinson et al. (Robinson, 1993, supra) for avians could reflect any of a large number of factors that can influence the efficiency of expression of antigen genes and/or the immunogenicity of DNA vaccines. Examples of such factors include the reproducibility of inoculation, construction of the plasmid vector, choice of the promoter used to drive antigen gene expression and stability of the inserted gene in the plasmid. Depending on their origin, promoters differ in tissue specificity and efficiency in initiating mRNA synthesis Xiang et al., Virology, 209:564-579 (1994)!, Chapman et al., Nucle. Acids. Res., 19:3979-3986 (1991)!. To date, most DNA vaccines in mammalian systems have relied upon viral promoters derived from cytomegalovirus (CMV). These have had good efficiency in both muscle and skin inoculation in a number of mammalian species but may not be as effective in avians. Another factor known to affect the immune response elicited by DNA immunization is the method of DNA delivery; parenteral routes can yield low rates of gene transfer and produce considerable variability of gene expression (Montgomery, 1993, supra). High-velocity inoculation of plasmids, using a gene-gun, enhanced the immune responses of mice (Fynan, 1993B, supra), Eisenbraun et al., DNA Cell Biol., 12: 791-797 (1993)!, presumably because of a greater efficiency of DNA transfection and more effective antigen presentation by dendritic cells. In any case, the corresponding state of current avian vaccine methodology can be improved. Indeed, there are no influenza virus vaccines for avians that are approved for use in the United States.
Pathogenic H5 and H7 subtypes of avian influenza virus emerge at irregular intervals and can decimate dense populations of poultry Easterday et al., Iowa State Univ. Press, 532-551 (1991)!. The feasibility of using a vaccine to prevent the spread of the virus will depend on the availability of preparations that induce durable protective immunity. Although available for restricted use, inactivated virus based vaccines are not standardized in terms of HA content Wood et al., Avian Dis., 29:867-872 (1985)! and generate antibodies to cross-reactive antigens such as nucleoprotein, thereby precluding detection of live virus in the flock. Such a situation prevents the determination of infection before symptoms become apparent, thus significantly hampering any attempt to isolate or destroy infected birds before the disease spreads throughout the flock. Similarly, the use of an another live virus, such as the fowlpox virus, to express an influenza antigen (Ramshaw et al., in "Molecular Approaches To The Control Of Infectious Diseases" Cold Spring Harbor Oct. 5-Oct. 9, 1994 pg. 116, Abstract) results in the infection of the avian flock with that live virus, an alternative that is, for the foreseeable future, unacceptable both in the United States and abroad. Finally, voluntary destruction of entire flocks, as practiced in the past in the United States Halvorsen et al., University of Wisconsin, 33-42 (1992)!, can effectively control avian influenza virus infection but only at a great cost. Such solutions are devastating to individual poultry farmers and can best be labeled as an archaic, stop-gap measure.
The last outbreak of highly pathogenic H5N2 avian influenza in the United States occurred in domestic chickens and turkeys in Pennsylvania in 1983-84, with devastating effects on the poultry industry Bean et al., J. Virol., 54: 151-160 (1985)!. The virus was eventually eradicated by quarantine and extermination of over 17 million birds at a direct cost of over 60 million dollars and an indirect cost to the industry of more than 250 million dollars Horimoto et al., Virol., 213: 223-230 (1995)!. Furthermore, this strategy is not even considered tenable under conditions of widespread infection of avian stocks such as is the case in Mexico and other developing countries. The pathogenic avian virus, A/Chick/Queretaro/95 (H5N2) for example, still persists in Mexico and could spread rapidly to the United States, necessitating some practical means to prevent the spread of virus in this country, as well as abroad. Eradication of the virus by destruction of infected birds in Mexico is not considered a reasonable option by the poultry industry. Therefore, an improved, more effective, and most importantly, reliable avian vaccine that does not contain an inactivated influenza virus, nor any other live or attenuated virus, is required in order to protect commercially important avians against pathogenic influenza viruses. It is in part to the attainment of this objective that the present invention is directed.